BMB Rep. 2019; 52(6): 391-396 BMB www.bmbreports.org Reports

CCAAT/enhancer-binding beta (C/EBP) is an important mediator of 1,25 dihydroxyvitamin D3 (1,25D3)-induced activator of nuclear factor kappa-B ligand (RANKL) expression in osteoblasts

Sungsin Jo1, Yun Young Lee2, Jinil Han3, Young Lim Lee1, Subin Yoon1,4, Jaehyun Lee1,4, Younseo Oh1, Joong-Soo Han5, Il-Hoon Sung6, Ye-Soo Park7,* & Tae-Hwan Kim1,* 1Hanyang University Hospital for Rheumatic Diseases, Seoul 04763, 2Department of Biomedical Sciences, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, 3Gencurix, Inc, Seoul 08394, 4Department of Translational Medicine, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, 5Biomedical Research Institute and Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul 04763, 6Department of Orthopedic Surgery, Hanyang University Hospital, Seoul 04763, 7Department of Orthopedic Surgery, Hanyang University Hospital, Guri 11923, Korea

Receptor activator of nuclear factor kappa B ligand (RANKL) which is marked by coordinated activities between osteoclasts expression in osteoblasts is regulated by 1,25-dihydroxy- and osteoblasts (1). Osteoclasts, multinucleated cells derived vitamin D3 (1,25D3). CCAAT/enhancer-binding protein beta from precursor monocyte lineages, play a critical role in bone (C/EBP) has been proposed to function as a transcription resorption (2). Two molecules are necessary for mature factor and upregulate RANKL expression, but it is still osteoclasts: macrophage colony-stimulating factor (M-CSF) and uncertain how C/EBP is involved in 1,25D3-induced RANKL receptor activator of NF-B ligand (RANKL) (3). RANKL expression of osteoblasts. 1,25D3 stimulation increased the expression in osteoblasts is critical for maintenance and expression of RANKL and C/EPB in osteoblasts and coordination of the osteoblast-osteoclast process (4). Osteoblasts enhanced phosphorylation and stability of these . induce expression of RANKL through various cytokines and Moreover, induction of RANKL expression by 1,25D3 in hormones. In particular, vitamin D3 is the key hormone that osteoblasts was downregulated upon knockdown of C/EBP. promotes osteoblastic activity and bone formation (5). In contrast, C/EBP overexpression directly upregulated 1,25-Dihydroxyvitamin D3 (1,25D3), a vitamin D3 active RANKL promoter activity and exhibited a synergistic effect on metabolite, is known to induce expression of RANKL in 1,25D3-induced RANKL expression. In particular, 1,25D3 osteoblasts and stromal cells through a treatment of osteoblasts increased C/EBP protein binding to (VDR) (6, 7). In RANKL regulation, vitamin D-responsive the RANKL promoter. In conclusion, C/EBP is required for element (VDRE) and RUNX2 are responsible for the promoter induction of RANKL by 1,25D3. [BMB Reports 2019; 52(6): located at the distal and proximal region, respectively (8-11). It 391-396] is well known that 1,25D3 increases RANKL expression, but RUNX2 is more controversial since there are reports that it does not contribute to RANKL expression (12, 13). Thus, there INTRODUCTION may be other candidate genes that respond to 1,25D3, and their function can be regulated for RANKL expression. Bone constantly cycles through formation and absorption, 1,25D3 induces CCAAT/enhancer-binding protein beta (C/EBP) via its binding site in the proximal region of RANKL *Corresponding authors. Tae-Hwan Kim, Tel: +82-2-2290-9245; (14, 15). Although the upregulation of RANKL by 1,25D3 has Fax: +82-2-2298-8231; E-mail: [email protected]; Ye-Soo Park, been intensively investigated, the relationship between the Tel: +82-31-560-2181; Fax: +82-31-560-8781; E-mail: hyparkys@ RANKL and C/EBP activation by 1,25D3 and the functional hanyang.ac.kr role of C/EBP in osteoblasts are not fully understood. Here, we demonstrate that C/EBP, which is responsive to https://doi.org/10.5483/BMBRep.2019.52.6.166 1,25D3, potentiates RANKL expression. By overexpressing and Received 20 July 2018, Revised 17 August 2018, knocking down C/EBP, we show that modulation of the Accepted 10 October 2018 directly contributes to activation and expression of the RANKL promoter. Keywords: 1,25-dihydroxyvitamin D3 (1,25D3), C/EBP, Osteo- blasts, RANKL

ISSN: 1976-670X (electronic edition) Copyright ⓒ 2019 by the The Korean Society for Biochemistry and Molecular Biology This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/li- censes/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Role of C/EBP in 1,25D3-induced RANKL expression Sungsin Jo, et al.

RESULTS viability or toxicity between 0 and 20 nM doses of 1,25D3, but these effect were observed at a higher dose of 50 nM 1,25D3 significantly increases VDR and C/EBP gene (Suppl. Fig. 2A and B). In this situation, ALP activity was expression in murine osteoblasts increased by 1,25D3 in a dose-dependent manner in MG63 High-throughput data with the accession number GSE51515 osteoblasts but decreased with 20 nM 1,25D3 in SaOS2 were downloaded from Omnibus (GEO) and osteoblasts (Suppl. Fig. 2C). Therefore, we selected analyzed. Using a publicly available microarray dataset, we concentrations of 10 and 20 nM for activation of investigated gene expression alterations after treatment with osteoblast-related gene promoters. Luciferase activities of the 10−7 M 1,25D3 for 24 hours. To assign a putative functional known osteoblast-specific ALP, OSE, OCN, and BSP promoters change in bone development, genes whose GO terms were were significantly elevated 2-3 fold by 20 nM 1,25D3 related to bone development were selected. As a result, 122 treatment (Suppl. Fig. 2D). Consistent with previous reports, genes were identified as bone development-related genes addition of 1,25D3 under osteogenic stimuli resulted in (Suppl. Fig. 1). Among them, 17 genes (NPR3, COL5A2, increased osteoblastic differentiation, as shown with ALP and DDX5, PTK2, FBN1, FST, GNAQ, HOXA10, FOXC1, PIAS2, Alizarin Red (ARS) staining (Suppl. Fig. 3). These data confirm VDR, RUNX1, C/EBP, SMAD3, SERP1, ASH1L, and RASSF2) that 1,25D3 has a potent effect on promoting osteoblast were upregulated when the POBs were treated with 1,25D3, differentiation. and 6 genes (GDF4, RUNX2, EPHA2, MMP9, UCMA, and USP1) were downregulated (Fig. 1). RANKL, also known as 1,25D3 induces RANKL expression via regulation of C/EBP TNFSF11, was upregulated, but it was not statistically As shown in Fig. 2A, increases in CYP24A mRNA and VD3R significant (indicated by the red arrow in Suppl. Fig. 1). We protein levels were affected by 1,25D3 treatment. In this noted that 1,25D3 resulted in increases in C/EBP and RANKL gene expression.

1,25D3 accelerates osteoblastic differentiation To investigate cellular effects of 1,25D3 on osteoblasts, we evaluated cell viability and toxicity in both MG63 and SaOS2 osteoblasts that were treated with various concentrations of 1,25D3 for 3 days. There was no significant difference in cell

Fig. 2. 1,25D3 induces RANKL expression via regulation of C/EBP. (A) Both MG63 and SaOS2 cells were treated with 1,25D3 as indicated, followed by immunoblotting (upper panel) and qRT-PCR (lower panel) (n = 5). (B) The cells were stimulated with 20 nM 1,25D3 for 24 h, followed by immunofluorescence using RANKL and C/EBP (n = 3). Scale bar Fig. 1. 1,25D3 significantly increases VDR and C/EBP gene is 50 m. (C) The cells were transfected with siRNA against expression in murine osteoblasts. Using microarray datasets, we C/EBP and control (CON) using Lipo3000, incubated for 48 h, investigated gene expression changes in response to a treatment and stimulated with 20 nM 1,25D3 for 24 h. The stimulated cells of 10−7 M 1,25D3 for 24 h. In order to assign putative were subjected to immunoblotting (upper panel) and qRT-PCR functional changes in bone development, genes whose GO terms (lower panel) (n = 5). (D) The cells were transfected with were related to bone development were selected. In total, 122 indicated deletion mutants of RANKL promoter, incubated for 48 genes were identified as bone development-related. A heat map h, treated with 1,25D3 for 24 h, and then analyzed with of bone development-related genes in POB cells treated with luciferase assay (n = 4). The Mann-Whitney U test was vehicle or 1,25D3. Blue, low expression; red, high expression. performed to determine statistical significance. Data are presented Among them, 24 genes with RANKL (marked in black) were as mean ± SD. P values indicate significant differences between differentially expressed. two groups. *P ＜ 0.05.

392 BMB Reports http://bmbreports.org Role of C/EBP in 1,25D3-induced RANKL expression Sungsin Jo, et al.

situation, we observed that expression of RANKL and C/EBP change in VD3R protein (Fig. 3B). We next used the inhibitors was elevated in both MG63 and SaOS2 cells when treated cycloheximide (CHX) to inhibit protein synthesis and MG132 with 1,25D3 in a time- and dose-dependent manners (Fig. 2A to inhibit the proteasome in order to observe changes at the and Suppl. Fig. 4). ALP, OCN, C/EBP, and RANKL were protein level. VD3R, C/EBP, and RANKL proteins were increased in osteoblasts treated with 1,25D3 (Suppl. Fig. 5 and reduced in the vehicle, whereas these proteins were only Fig. 2B). Intriguingly, upregulation of RANKL expression in modestly reduced in response to 1,25D3 (Fig. 3C). Moreover, response to 1,25D3 was attenuated by the C/EBP knockdown 1,25D3-induced VD3R, C/EBP, and RANKL proteins were at the mRNA and protein levels (Fig. 2C). In addition, 1,25D3 degraded by the proteasome pathway (Fig. 3D). These results treatment activated the RANKL promoter (approximately 2Kb) suggest that 1,25D3 enhances the phosphorylation and in both osteoblast cell lines (Suppl. Fig. 6), but showed stability of C/EBP protein. relatively higher response in the promoter within 1Kb (Fig. 2D). Therefore, 1,25D3 induces RANKL expression by 1,25D3-induced upregulation of C/EBP contributes to regulating C/EBP. RANKL expression in osteoblasts To extend on the above findings, we speculated that 1,25D3 1,25D3 enhances phosphorylation and stability of the C/EBP might positively regulate changes in RANKL expression via protein C/EBP. Overexpression of C/EBP in combination with To further assess the regulation of the C/EBP protein by 1,25D3 stimulation exhibited a synergistic effect on RANKL 1,25D3 in osteoblasts, we stimulated osteoblasts with various expression in comparison to either 1,25D3 or C/EBP alone concentrations of 1,25D3 for 6 h and then analyzed (Fig. 4A and B). C/EBP overexpression markedly induced phosphorylation of p38 and ERK protein, two 1,25D3 RANKL promoter activity (Suppl. Fig. 7) and its expression was responsive kinases. 1,25D3 induced the phosphorylation of quantified by qRT-PCR (Suppl. Fig. 7, lower panel). C/EBP p38, ERK, and C/EBP proteins in a dose-dependent manner overexpression significantly also induced the proximal region (Fig. 3A). We also used SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, to confirm an effect on the phosphorylated C/EBP protein. The two inhibitors prevented C/EBP-Thr235 phosphorylation through 1,25D3 without a

Fig. 4. 1,25D3-induced upregulation of C/EBP contributes to RANKL expression in osteoblasts. Both MG63 and SaOS2 cells were transduced with C/EBP (2.5 g) or empty vector for 48 h, Fig. 3. 1,25D3 enhances C/EBP protein phosphorylation and treated with 1,25D3 for 24 h, and then analyzed by (A) stability. (A) Both MG63and SaOS2 cells were stimulated with immunoblotting (n = 5) or (B) qRT-PCR (n = 5). (C) Indicated 1,25D3 as indicated for 6 h. (B) The cells were pre-treated with deletion mutants of RANKL promoter was transiently co-transfected SB203580 or PD98059 for 5 min and stimulated with 20 nM with C/EBP (2.5 g) or empty vector in both MG63 and SaOS2 1,25D3 for 6 h. (C) The cells were pre-treated with vehicle-alone cells. The transfected cells were incubated for 48 h and then (Ethanol) or 20 nM 1,25D3 for 1 day and harvested after analyzed using a luciferase assay (n = 4). (D) Cells were stimulation with 20 g/ml cyclohexamide (CHX) at the indicated stimulated with 20 nM 1,25D3 for 24 h and then analyzed with times. (D) The cells were pre-treated with MG132 for 5 min and a chromatin immunoprecipitation (ChIP) assay using the C/EBP stimulated with 20 nM 1,25D3 for 6 h. All samples were antibody (MG63, n = 4; SaOS2, n = 4). The Mann-Whitney U subjected to immunoblotting. All experiments were carried out at test was performed to determine statistical significance. Data are least four times, and data consistency was observed between presented as mean ± SD. P values indicate significant differences experiments. Representative images are shown. between two groups. *P ＜0.05. http://bmbreports.org BMB Reports 393 Role of C/EBP in 1,25D3-induced RANKL expression Sungsin Jo, et al.

(less than 1Kb) compared to a 2Kb promoter region of human RANKL in osteoblasts and its progenitor cells. In this study, RANKL gene (Fig. 4C). Interestingly, #1 of sites of three 1,25D3 treatment in osteoblasts specifically induced putative C/EBP binding sites on the proximal RANKL upregulation of RANKL expression without changes in OPG promoter was selectively enhanced in response to mRNA (Fig. 2B). We tested secreted RANKL from the culture 1,25D3-induced C/EBP protein (Fig. 4D). Taken together, supernatant using an enzyme-linked immunosorbent assay these findings indicate that 1,25D3-induced upregulation of (ELISA), and there was no detectable soluble RANKL in C/EBP contributes to RANKL expression in osteoblasts. response to 1,25D3 (data not shown). There are two possible explanations for this. 1,25D3-dependent induction of RANKL DISCUSSION in osteoblasts might produce either a transmembrane form to contact and interact with osteoclast precursors for osteoclast In this study, we sought to identify the signaling pathways and activation or intracellular form to accumulate in cells. transcription factors involved in stimulation of Previous studies have revealed many putative binding sites 1,25D3-induced C/EBP and RANKL gene expression. We in the human RANKL promoter (8): C/EBP, VDRE, heat shock observed that 1,25D3 results in p38- and ERK-dependent factor 2 (HSF2), cAMP-responsive element-binding protein activation and phosphorylation of C/BEP in both MG63 and (CREB), runt-related 2 (RUNX2), and SaOS2 osteoblasts. Furthermore, C/EBP knockdown slightly nuclear factor-erythroid-derived 2 (NF-E2). We analyzed reduced RANKL expression and attenuated 1,25D3-induced whether either the C/EBP or RUNX2 gene was modulated in the gene level. In contrast, 1,25D3 and C/EBP overexpression the presence of 1,25D3. Interestingly, the cells responded to had a synergistic effect on RANKL expression. Finally, we 1,25D3 by increasing C/EBP expression and decreasing demonstrated that, in response to 1,25D3, C/EBP binds RUNX2 gene expression, as shown in the microarray data in directly to the RANKL promoter in osteoblasts. Fig. 1. We compared luciferase activity of the RANKL Vitamin D is an important regulator of bone mineralization promoter to the expression of the RUNX2 and C/EBP genes. and metabolism. 1,25D3 is the most active metabolite of As expected, the RANKL promoter was dramatically induced vitamin D3, with high affinity for nuclear VDR (16). in transduction of the C/EBP gene than RUNX2, indicating Hydroxylation of vitamin D metabolites at D-24 hydroxylase that C/EBP is an essential factor for RANKL expression (data (CYP24A1) is the first step in metabolite inactivation and not shown). excretion (17). Basal expression of CYP24A1 is usually low, We provided evidence of a critical role for 1,25D3 in but 1,25D3 strongly induces CYP24A1 gene expression (18). mediating the phosphorylation and stabilization of C/EBP, Consistent with previous results, we observed increases in which facilitates RANKL gene upregulation, and contributes to VD3R protein and CYP24A1 mRNA levels in response to our understanding of RANKL expression in osteoblasts. 1,25D3 (Fig. 2A and B). It is well known that C/EBP is a critical factor during MATERIALS AND METHODS adipocyte and chondrocyte differentiation (19). C/EBP is an important indicator of differentiation and control of target gene Cell lines, chemicals, and plasmids transcription. Despite the fact that C/EBP is necessary for Both MG63 and SaOS2 osteoblast cell lines were obtained osteoblastic activity and bone formation via RUNX2 and ATF4 from Heekyoung Chung (Department of Pathology, College of regulation (20), there are fewer reports on C/EBP function in Medicine, Hanyang University) and Korean Cell Line Bank osteoblasts than in other cell lineages. Our previous work has (Seoul, Korea). MG63 and SaOS2 were grown in high-glucose revealed that RUNX2 and C/EBP proteins in bone-derived DMEM medium (Hyclone, SH30243.01) and RPMI1640 cells cooperate and promote IL-23 expression in ankylosing (Hyclone, SH30027.01) supplemented with 10% fetal bovine spondylitis (AS) (21). In addition, inflammatory cytokines in AS serum (FBS) (Gibco, 10082-147) and 1× antibiotics (Gibco, patient sera stimulate RUNX2 and C/EBP proteins via 15140-122). 1,25D3 (Sigma-Aldrich, D1530) was dissolved in osteogenic induction (22, 23). In this study, we propose that absolute ethanol, respectively. For in vitro drug treatment, induction of the RANKL gene by 1,25D3 is mediated by 1,25D3 was prepared as a 20 M stock solution and diluted in C/EBP. We therefore believe that the function of the C/EBP fresh medium at the indicated concentrations. SB203580 gene is a crucial factor in osteoblasts and in bone research. (559398) and PD98059 (513001) were purchased from Merck Molecular RANKL and OPG play key roles in regulating Millipore, and DMSO was used as the vehicle. physiological and pathological bone homeostasis. In general, Osteoblast-related promoters of alkaline phosphatase (ALP), RANKL can be expressed in three different molecular forms osteocalcin (OCN), osteoblast-specific elements (OSE), and consisting either of (1) a membrane-bound form, (2) a secreted bone sialoprotein (BSP) were provided by Dr. Kwang Youl Lee form, or (3) an intercellular form. It has been reported that (College of Pharmacy, Chonnam National University, soluble RANKL is produced by B cells (24), neutrophils (25), Gwangju, Korea) (28). C/EBP and the empty vector were activated T cells (26), synoviocytes (27), and osteoblasts (4). generously provided by Dr. Yung Jong Lee (Division of Relatively less is known about the molecular mechanism of Rheumatology, Department of Internal Medicine, Seoul

394 BMB Reports http://bmbreports.org Role of C/EBP in 1,25D3-induced RANKL expression Sungsin Jo, et al.

National University Bundang Hospital) (29). The RANKL ACKNOWLEDGEMENTS promoter was generously provided by Dr. Sakamuri V. Reddy (Department of Pediatrics, Medical University of South We thank Dr. Sung Eun Wang (Hanyang Biomedical Research Carolina) (30). siRNA was obtained from Genolution (Seoul, Institute, Hanyang University) for technical assistance regarding Korea). siRNA information is given in Supplementary Table 3. the ChIP assay. Immunofluorescence images were analyzed by a confocal microscope at Hanyang LINC Analytical Equipment Gene Expression Omnibus (GEO) analysis Center (Seoul). Gene expression datasets were downloaded from the NCBI This work was supported by the Basic Science Research Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih. Program through the National Research Foundation of Korea gov/geo) under the accession numbers GSE41954 and (NRF) funded by the Ministry of Science, ICT, & Future GSE51514 (31). MC3T3-E1 cells (pre-osteoblasts, POBs) were (NRF-2016R1A2B4008606) and the Ministry of Education treated with vehicle or 10−7 M 1,25D3 for 24 hours. RNA was (2017R1A6A3A11034394). The study was also supported by a then isolated and applied to gene expression microarrays. The Korea Health Technology R&D grant through the Korea Health microarray experiments were conducted in biological Industry Development Institute (KHIDI), which is funded by triplicate. Differentially expressed genes were assessed by a the Ministry of Health & Welfare, Republic of Korea linear regression method using the R/Bioconductor limma (HI17C0888). package (32). An adjusted P-value ＜ 0.05 was considered statistically significant. All microarray analyses and visualiza- CONFLICTS OF INTEREST tion were conducted using R 3.4.1 (www.r-project.org). The authors have no conflicting interests. 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